Laboratory of Microbial Metabolic Potential.

In the Laboratory of Microbial Metabolic Potential, we are exploring the hidden gifts and talents of the microorganisms and applying them in industrial processes.

About the Laboratory of Microbial Metabolic Potential (Onaka Lab.)

The Laboratory of Microbial Metabolic Potential is an IFO-endowed course at the Graduate School of Agricultural and Life Sciences, the University of Tokyo, since 2012. The IFO, the Institution for Fermentation, Osaka, is a public interest incorporated foundation in Japan, and it provides grants for microbial researches that contribute to the further development of microbiology.

“Microorganism” is a generic term for tiny invisible organisms. These ubiquitous organisms are soil decomposers, and are involved in maintaining global environment such as the carbon and nitrogen cycles. In addition, they are normal inhabitants in the human body, pathogens for mammals and plants, and producers of a wide variety of organic materials. However, usually, we do not mind their presence in our daily life.

The recent advances in microbial genome analysis have changed the perception of microorganisms. Microbial genomic information revealed that microorganisms have biological diversity far beyond that between mammals and plants, and we are expecting to discover novel microbial functions from these genomic data.

In the Laboratory of Microbial Metabolic Potential, we are exploring the hidden gifts and talents of the microorganisms and applying them in industrial processes. In particular, we would be focusing on actinomycetes, soil bacteria known for their ability to produce antibiotics, and on identifying novel potent biosynthetic machinery in their secondary metabolism.

The current research interests of our laboratory are (i) development of drug screening from natural compounds with combined bacterial culture, (ii) biosynthesis of Goadsporin, which is a liner peptide including thiazole and oxazole rings, (iii) indolocarbazole biosynthesis.

Our Research

The development of new drugs relies heavily on the discovery of novel natural products produced by microorganisms. The recent genomic analysis of some Streptomyces strains has revealed the presence of biosynthetic gene clusters for about 30 secondary metabolites; these data imply that a single Streptomyces strain can produce over 30 secondary metabolites. However, some of these secondary-metabolite genes are cryptic in fermentation culture. Because many biosynthetic genes for secondary metabolites remain silent under normal laboratory culture conditions, the valuable secondary metabolites encoded by them are not studied.

We found that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the physical contact between the mycolic-acid-containing bacteria and Streptomyces. The production of red pigment by Streptomyces lividans TK23 was induced by co-culture with Tsukamurella pulmonis TP-B0596, however there is no pigment when these two bacterium were partitioned. (Fig. 1-1)

Observation of combined-culture by scanning electron microscopy (SEM) indicated that adhesion of live MACB to S. lividans mycelia were a significant interaction that resulted in formation of co-aggregation. (Fig. 1-2)

We used these results to develop a new co-culture method, called 'combined culture' method, which facilitates the screening of natural products. To date, we have already discovered 5 types, 17 novel compounds with combined-culture. (Fig. 1-3) (Fig. 1-4)

Fig. 1-1 : The stimulation of mycolic acid containing bacteria were directly transmitted by physical contact. When Streptomyces and mycolic acid-containing bacteria were grown in a dialysis flask, which contains two compartments to grow both bacteria separately through a dialysis membrane, S. lividans did not produce red pigments. (ref. 1-1)
Red pigment production was only detected in the border of both bacteria, suggesting that physical contact triggers to produce the red pigments. (ref. 1-1)

(2) Biosynthesis and genetic engineering of goadsporin, a liner azole-containing peptide produced by Streptomyces sp. TP-A0584

With the progress of genome-mining techniques for secondary metabolite screening, many kinds of a liner azole-containing peptide (LAPs) synthesized by ribosome were discovered from a wide variety of microorganisms. The common feature of LAPs is the peptide backbone containing thiazole, metylthiazole, and oxazole rings, which are derived from serine, threonine, and cycteine, respectively, and these heterocyles contribute for the bioactivity and the structure stability.

LAPs biosynthetic gene clusters consist of the structure gene, the post-translational-modificaion enzyme genes, transcriptional regulator genes, and the immunity genes. Interestingly, although the all of them contains enzymatic genes for thiazole or oxazole formation, these genes have very low similarities among these producing strains.

Over 50 GS analogs were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is applied for heterocyclization of peptide. This approach will open the door to biosynthesize the new biological active peptides.

(3) Genome mining reveals a minimum gene set for the biosynthesis of 32-membered macrocyclic thiopeptides, lactazoles.

Thiopeptides are produced mainly by actinomycetes and typically contain highly modified sulfur-containing peptides, which have a characteristic macrocycle knotted with pyridine or piperidine, a six-membered nitrogen-containing ring. Although more than 100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We used genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles from Streptomyces lactacystinaeus OM-6519. To date, the ring size of macrocyclic thiopeptide is limited to 26, 29, or 35 atoms, while lactazoles are structurally novel thiopeptides with a 32-membered macrocycle (Fig. 3-1). The 2-oxazolyl-6-thiazolylpyridine core with the 3-position connected to tryptophan through an amino linkage also provides a unique structure in thiopeptides. Lactazoles did not show any antimicrobial activities, however, we found that inhibitory activities for the bone morphogenetic protein (BMP) signal cascade in vivo, which could add to a new aspect of therapeutic treatment against to thiopeptide family antibiotics.

Lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). It is the smallest cluster among the known thiopeptide biosynthetic gene clusters (Fig. 3-2). The structure gene, lazA contains the precursor peptide sequence, and it is classified into a phylogenetically distinct clade. lazC is involved in the macrocyclization process, leading to central pyridine moiety formation by gene disruption.

Substitution of the endogenous promoter with that of godA, a gene involved in goadsporin biosynthesis results in an approximately 30-fold increase in lactazole A production. A using the godA promoter to regulate the lactazole biosynthetic machinery, production of two analogs, S11C and W2S, was achieved.

We expect that this compact biosynthetic machinery has high potency to lend large diversity to the thiopeptide core structures. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development.

Indolocarbazoles are compounds with a 6-ring-fused plane structure, which mimics ATP. Therefore, compounds containing an indolocarbazole core could be used to inhibit protein kinases such as Protein Kinase A (PKA) and Protein Kinase C (PKC), and are potential candidates of antitumor drugs. Staurosporine, an indolocarbazole compound, is biosynthesized by Streptomyces sp. TP-A0274. A whole set of biosynthetic genes have been already cloned, and these studies revealed that 4 enzymes, StaO, StaD, StaP, and StaC, are responsible for indolocarbazole core biosynthesis. The starter unit for the indolocarbazole core is 2 molecules of tryptophane. In the biosynthesis, StaO that encodes a monooxygenase, catalyzes the conversion from tryptophane to the indolepyruvic acid imine form (IPA imine). StaD then catalyzes the intermolecular-coupling reaction of 2 IPA imines to give a chromopyrrolic acid (CPA), which is a key intermediate of indolocarbazoles. Finally, intramolecular C-C bonds are formed between indole rings, followed by oxidation of the pyrrole moiety by StaP and StaC to yield K252c, an indolocarbazole core.

StaP, the C-C coupling enzyme, is a cytochrome P450. The X-ray crystal structures of CPA-bound and free forms of StaP demonstrate the molecular mechanism of substrate recognition by StaP. StaP is a typical P450 fold; however, it catalyzes the reaction by means of an indole cation radical intermediate, which is similar to the peroxidase reaction.